Abstract
In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at -177/-171 and -191/-187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas.
Highlights
There is consistent data indicating that in human stomach as well as in other organs mucin genes are expressed in a ʈ Recipient of a CHRU de Lille-Region Nord-Pas de Calais Ph.D. fellowship
Expression of MUC2, Cdx-1, and Cdx-2 in Human Gastric and Colon Carcinoma Cell Lines—Expression of MUC2, Cdx-1, and Cdx-2 was studied by RT-PCR
In the current study we demonstrate the direct involvement of the Cdx-2 homeodomain protein in the transcriptional regulation of the MUC2 gene in gastric and colon carcinoma cells
Summary
Cdx-1, caudal-related homeobox factor 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SIF1, sucraseisomaltase footprint 1; 5Ј-UTR, 5Ј-untranslated region; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase. For EMSAs, sense and antisense oligonucleotides were annealed to produce double-stranded DNA. Positions of the Cdx-2 binding sites relative to MUC2 transcription start site are indicated [36]. GGG TGC AAT AAA ACT TTA TGA GTA GAA GGC TGC GTT TAC CCA GGG AGC CAT AAA GAG ATG ACC T GAA GGC TGC GGC GTG CCA GGG AGC CGC GTG GAG ATG ACC T tion as transcriptional regulators of MUC2. The implications in intestinal metaplasia and gastrointestinal cell differentiation are discussed
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